This invention relates to novel amine-containing compounds useful for therapeutics and methods of making and using the same.
It is well known that most of the bodily states in mammals including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man. Classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, intracellular RNA. These interactions involved the binding of complementary xe2x80x9cantisensexe2x80x9d oligonucleotides or their analogs to the transcellular RNA in a sequence specific fashion such as by Watson-Crick base pairing interactions.
The pharmacological activity of antisense compounds, as well as other therapeutics, depends on a number of factors that influence the effective concentration of these agents at specific intracellular targets. One important factor is the ability of antisense compounds to traverse the plasma membrane of specific cells involved in the disease process.
Cellular membranes consist of lipid protein bilayers that are freely permeable to small, nonionic, lipophilic compounds and inherently impermeable to most natural metabolites and therapeutic agents. Wilson, Ann. Rev. Biochem. 1978, 47, 933. The biological and antiviral effects of natural and modified oligonucleotides in cultured mammalian cells have been well documented, so it appears that these agents can penetrate membranes to reach their intracellular targets. Uptake of antisense compounds into a variety of mammalian cells, including HL-60, Syrian Hamster fibroblast, U937, L929, CV-1, and ATH8 cells has been studied using natural oligonucleotides and nuclease resistant analogs, such as alkyl triesters, Miller, et al., Biochemistry 1977, 16, 1988; methylphosphonates, Marcus-Sekura, et al., Nuc. Acids Res. 1987, 15, 5749 and Miller, et al., Biochemistry 1981, 20, 1874; and phosphorothioates, Ceruzzi, et al., Nucleosides and Nucleotides 1989, 8, 815; Miller, et al., Biochemistry 1987, 16, 1988; and Loke, et al., Curr. Top. Microbiol. Immunol. 1988, 141, 282.
Enhanced cellular uptake has previously been achieved by attachment of functional groups to the 3xe2x80x2 and 5xe2x80x2 end of oligonucleotides to enhance cellular uptake in specific cell types. Previous studies have shown that plasmid DNA completed with an (asialo)glycoprotein-poly(L-lysine) conjugate, could be targeted to hepatocytes, which contain unique cell surface receptors for galactose-terminal (asialo)glycoproteins. Wu, et al., Biochemistry 1988, 27, 887. Other groups have synthesized oligodeoxyribonucleotides that have a 5xe2x80x2-attached alkylating agent and a 3xe2x80x2 attached cholesterol moiety and determined that these modified oligonucleotides were taken up into cells more efficiently than control compounds without the steroid moiety. Zon, G. in Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression 234-247, ed. J. S. Cohen (CRC Press, Boca Raton Fla., 1989). Letsinger, et al., Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 653, have also synthesized cholesteryl-conjugated phosphorothioates whose anti-HIV activity is significantly greater than natural oligonucleotides with the same sequence. Additional modifications include conjugation of oligonucleotides to poly(L-lysine) alone. Stevenson, et al., J. Gen. Virol 1989, 70, 2673 and Lemaitre, et al., Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 648. This modification enhanced the antiviral activity of the compound studied presumably due to increased cellular uptake imparted by the polycationic poly(L-lysine).
The conjugation of polyamines to oligonucleotides have been found to enhance cellular uptake of oligonucleotides, increased lipophilicity, cause greater cellular retention and increased distribution of the compound. Vasseur, Nucleosides and Nucleotides 1991, 10, 107 prepared abasic sites at different sites of oligothymidylates by acid hydrolysis. Thereafter the abasic sites were functionalized with functionalities such as 3-amino carbazole, 9-amino elipticine and psoralen. Vasseur, et al., also refer to unpublished results in which the functionalities spermidine and proflavin were employed. The abasic site was generated by one of the following three methods: (i) selective depurination by acid treatment in a pyrimidine-rich oligonucleotide having one purine in a chosen site, (ii) incorporating 2xe2x80x2,3xe2x80x2-dideoxynebularine at the 5xe2x80x2-end with the nebularine phosphoramidite at the last step of the oligonucleotide synthesis, and subsequent acid treatment (30 mM HCl at 37xc2x0 C.) to create an abasic site at 5xe2x80x2 end (in this case the open-chain structure is CHOxe2x80x94(CH2)2xe2x80x94CHOHxe2x80x94CH2Oxe2x80x94 at the 5xe2x80x2 end and the conjugate from the amine RNH2 is RNHxe2x80x94(CH2)3xe2x80x94CHOHxe2x80x94CH2xe2x80x94O-Oligo), and (iii) incorporating a protected abasic 2xe2x80x2-deoxy-D-ribofuranose nucleotide synthon that has a photo-labile O-nitrobenzyl group as the anomeric hydroxyl-protecting group in oligonucleotide synthesis and removing it prior to conjugation.
Groebke and Leumann used a silyl-protecting group at the anomeric center to generate the abasic site. 2xe2x80x2-Deoxy-5-O-dimethoxytrityl-D-ribofluranose was silylated at the 1-O-position using TBDMSCl and the silyl group was removed later by hydrolysis at pH 2.0 to yield the abasic site. Unfortunately, fluoride-ion-mediated deprotection of the silyl group caused a xcex2-elimination and DNA degradation.
McLaughlin""s group has utilized 1-(xcex2--D-2-deoxyribosyl)-2-pyrimidone-based phosphoramidite to generate abasic sites at pH3.0. The N-glycosyl cleavage occured, however, slower in oligonucleotides than in parent nucleosides; nearly 60 hours of acid treatment was necessary to generate 90% abasic site formation. However, conjugation chemistry via enzymatically generated abasic sites are unknown in the literature.
Le Doan, et al., Nucleic Acids Research 1987, 15, 8643 teaches oligothymidylates covalently linked to porphyrins at their 3xe2x80x2 end via one of the linkers xe2x80x94Oxe2x80x94CH2xe2x80x94COxe2x80x94NHxe2x80x94(CH2)2xe2x80x94NH or PO4xe2x80x94(CH2)6xe2x80x94NHxe2x80x94. Le Doan, et al., also used the linker PO4xe2x80x94(CH2)6xe2x80x94NHxe2x80x94 to link porphyrins to the 5xe2x80x2 end of oligothymidylates. Another group, Summerton, et al., U.S. Pat. No. 5,034,506 issued Jul. 23, 1991 teaches morpholino subunits, linked together by uncharged, achiral linkages such as amides. As described in PCT/US91/04086 filed Jun. 10, 1991, polyamines have also been linked at the 5xe2x80x2 end of an oligonucleotide at the 5xe2x80x2 site of the sugar moiety of the terminal nucleoside and at the 2-position carbon of the heterocyclic base of 2xe2x80x2-deoxyadenosine, 2xe2x80x2-deoxyguanosines and other purines and purine analogs by known procedures as described in PCT/US/91/00243 filed Jan. 11, 1991.
Novel amines and methods of preparing the same are greatly needed in order to enhance cellular uptake of oligonucleotides, increase lipophilicity, cause greater cellular retention and increase distribution of the compound within the cell. The present invention fulfills this need.
It is one object of the present invention to provide novel amine-containing compounds useful in therapeutics.
It is a further object of the present invention to provide methods of producing said novel compounds.
It is another object of the present invention to provide methods of modulating the production of a protein by an organism.
It is still a further object of the present invention to provide methods of treating a mammal suffering from a disease characterized by the undesired production of a protein.
It is yet a further object of the present invention to provide methods of diagnosing the presence of an RNA in a biological sample.
These and other objects will become apparent from the following description and accompanying claims.
The present invention provides compounds which may have enhanced efficacy as an antisense-based therapy. Compounds of the present invention can have enhanced cellular uptake, increased lipophilicity, cause greater cellular retention and demonstrate increased distribution. Furthermore the present invention provides simple methods for synthesis of these novel compounds.
In accordance with some embodiments of the present invention, compounds having the structure: 
wherein R1 and R2 are independently H, a nucleotide, oligonucleotide, or an amine-containing species, and at least one of R1 and R2 is a purine containing oligonucleotide, R3 is a linear or cyclic amine-containing species, and X is H, Oxe2x80x94R11, Sxe2x80x94R11, F, Cl, Br, CN, CF3, OCF3, OCN, SOCH3, SO2CH3, ONO2, N3, HN2, heterocylcoalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a reporter molecule, an RNA cleaving group, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide wherein R11 is H, C1 to C10 straight or branched chain lower alkyl or substituted lower alkyl, C2 to C10 straight or branched chain lower alkenyl or substituted lower alkenyl, C3 to C10 straight or branched chain lower alkynyl or substituted lower alkynyl, a 14C containing lower alkyl, lower alkenyl or lower alkynyl, C7 to C14 substituted or unsubstituted alkyaryl or aralkyl, a 14C containing C7 to C14 alkaryl or aralkyl, alicyclic, heterocyclic, a reporter molecule, a RNA cleaving group, a group for improving the pharmacokinetic properties of an oligonucleotide or a group for improving the pharmacodynamic properties of an oligonucleotide, are provided.
In accordance with still other embodiments of the present invention, compounds having the structure: 
wherein R4 is an oligonucleotide and M is a pendent group having an amine-containing species attached thereto are provided.
Methods of preparing such compounds utilizing enzymatic reagents are also provided in some aspects of the invention. Thus compounds of Formula I may be prepared by methods comprising the steps of providing a synthon having the structure: 
wherein R1 and R2 are independently H, a nucleotide, oligonucleotide or amine-containing species, and at least one of R1 and R2 is a purine containing oligonucleotide, and X is H, Oxe2x80x94R11, Sxe2x80x94R11, F, Cl, Br, CN, CF3, OCF3, OCN, SOCH3, SO2CH3, ONO2, N3, HN2, heterocylcoalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a reporter molecule, an RNA cleaving group, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide wherein R11 is H, C1 to C10 straight or branched chain lower alkyl or substituted lower alkyl, C2 to C10 straight or branched chain lower alkenyl or substituted lower alkenyl, C3 to C10 straight or branched chain lower alkynyl or substituted lower alkynyl, a 14C containing lower alkyl, lower alkenyl or lower alkynyl, C7 to C14 substituted or unsubstituted alkyaryl or aralkyl, a 14C containing C7 to C14 alkaryl or aralkyl, alicyclic, heterocyclic, a reporter molecule, a RNA cleaving group, a group for improving the pharmacokinetic properties of an oligonucleotide or a group for improving the pharmacodynamic properties of an oligonucleotide. Thereafter the synthon is reacted with R3, wherein R3 is a linear or cyclic amine-containing species, under reducing conditions to yield the final product.
Compounds of Formula II may also be prepared enzymatically by providing a starting material having the structure: 
wherein R4 is an oligonucleotide, R12 is an oligonucleotide and B is urea or a heterocyclic base having a corresponding glycosylase and reacting the starting material with an endonuclease to generate a conjugated xcex1,xcex2-unsaturated system in the sugar residue of the 3xe2x80x2 terminal nucleotide. Thereafter the compound having a conjugated xcex1,xcex2-unsaturated system is reacted with a pendent group containing a nucleophile functionality thereon. Following addition of the pendent group the double bond of the xcex1,xcex2 system is reduced with a reducing agent. An amine-containing species may then be attached to the pendent group via an alkylation reaction. Alternatively, an amine-containing species may be attached to a pendent group which is a bifunctional linker.
In accordance with still other embodiments of the present invention compounds having the structure: 
wherein R4 is an oligonucleotide, R5 is a linear or cyclic amine-containing species containing at least one non-amide nitrogen atom, and R6 is H, a purine heterocycle or a pyrimidine heterocycle, are provided. Methods of preparing compounds of Formula III are also provided in some aspects of the present invention comprising the steps of reacting an oligonucleotide having a 3xe2x80x2 ribofuranosyl sugar with an oxidizing agent to produce an activated dialdehyde-terminated oligonucleotide and reacting said activated oligonucleotide with a linear or cyclic amine-containing species under reducing conditions to yield said compound.
In accordance with other aspects of the invention compounds having the structure: 
wherein B is a purine or pyrimidine heterocyclic base, R8 and R9 are independently H, PO2xe2x88x92, a nucleotide, an oligonucleotide or an amine-containing species, and at least one of R8 and R9 is a purine containing oligonucleotide, and at least one of R8, R9 and A is a species comprising the formula L1xe2x80x94L2-polyamine wherein L1 is an amino linker and L2 is a heterobifunctional linker; and wherein if R8 is not a purine containing ligonucleotide or polyamine species, then R8 is a nucleotide or PO2xe2x88x92; if R9 is not a purine containing oligonucleotide or polyamine species, then R9 is H or a nucleotide; and if A is not a polyamine species then A is H or OH are provided.
Therapeutic and diagnostic methods are also encompassed by the present invention. Methods of modulating the production of protein by an organism comprising contacting an organism with a compound having the structure of Formula I, formula II, Formula III or Formula IV are encompassed by some embodiments of the present invention. In other aspects of the invention, methods of treating an animal having a disease characterized by undesired production of protein comprising contacting an animal with a compound having the structure of Formula I, Formula II, Formula III, or Formula IV in a pharmaceutically acceptable carrier are provided. Still other methods of the present invention provide methods for detecting the presence or absence of an RNA in a biological sample suspected of containing said RNA are provided comprising contacting a sample with a compound having the structure of Formula I, Formula II, Formula III or Formula IV wherein the compound is specifically hybridizable with the RNA and detecting the presence or absence of hybridization of the compound to the sample wherein hybridization is indicative of the presence of RNA in the sample.